1. Field of the Invention
This invention relates to apparatus and methods for collecting blood which is to be used in screening tests for activation of the coagulation system. Platelet Factor 4 (PF4) is a small molecular weight protein that is secreted from the .alpha.-granules of platelets when cells undergo the release reaction. This phenomenon occurs when the platelets become activated subsequent to contact with subendothelial tissue and a variety of other physiological agents including thrombin, adenosine diphosphate and epinephrine. PF4 in a blood sample is measured using classical radioimmunoassay techniques. Unfortunately classical blood collection techniques result in activatin of the release mechanism in the collected blood sample. Great care must be exercised to insure that the release reaction measured is a result of activating the release reaction in vivo and not in vitro.
2. Prior Art
The prior art discloses a wide variety of techniques and approaches for stabilizing collected blood samples against platelet release reactions.
Careful handling of blood samples is required to inhibit the platelet release reaction. Generally the blood is collected by venipuncture techniques using standard EDTA (ethylenediaminetetraacetic acid) containing vacuum blood collection containers. The blood is mixed with the EDTA in the container by gentle inversion and the container is placed in an ice bath. After thirty minutes the sample is centrifuged at 2500.times.g and 2.degree.-4.degree. C. for twenty minutes. Centrifugation must take place within two hours after collection. The plasma is separated and analyzed directly or it can be stored at 2.degree.-4.degree. C. for 24 hours or at -20.degree. C. for up to three months.
Thrombosis Research, 6, 543-548 (1975) describes collection of blook in the presence of EDTA and theophylline but requires centrifugation at 0.degree. C.
Thrombosis Research, 12, 851-861 (1978) describes the collection of blood for PF4 analysis in the presence of platelet release inhibitor EDTA, prostoglandin E, and theophylline. This procedure also requires inconvenient centrifugation at low temperature.
Niemetz, et al, Proc. Soc. Exp. Biol. Med, 128, 658 (1968) describes the effects of various anesthetics on clot retraction, adenosine diphosphate and thrombin-induced platelet aggregation. In those studies it was found that p-aminobenzoyldiethylaminoethanol hydrochloride (Procaine) and related homologs such as p-aminobenzoyldibutylaminoethanol (Butacaine) and p-butylaminobenzoyldimethylaminoethanol (Tetracaine) inhibited thrombin-induced platelet aggregation. Butacaine and tetracaine inhibited ADP-induced platelet aggregation while Procaine did not. Butacaine, tetracaine and procaine inhibited thrombin-induced platelet aggregation. Unexpectedly it has now been found that procaine and not butacaine and tetracaine inhibit the release of platelet factor 4 during the collection process.